The research described in this proposal is directed towards elucidating the mechanism by which secretory proteins are translocated across the endoplasmic reticulum. Particular emphasis will be placed upon i) characterization of a GTP-dependent translocation component, ii) further characterization of the signal peptidase complex and iii) investigating the mechanism of rebosome binding to the membrane. A GTP-dependent translocation factor has been detected which is essential for the formation of a functional ribosome-membrane junction. Functional studies of the GTP-dependent translocation factor will be continued to more succinctly define the factor's role in translocation of secretory proteins and integration of membrane proteins. The GTP-dependent translocation component will be identified in intact membranes and assayed in detergent solubilized fractions by photoaffinity labeling with (Alpha-32p)GTP. Purification of the GTP-dependent translocation factor can then be accomplished using standard methods for the isolation of integral membrane proteins. The membrane organization of the signal peptidase protein complex will be examined using subunit specific antibodies in conjunction with protein modification reagents. The signal peptidase complex will be tested in vitro for ribosome binding activity and signal peptide hydrolase activity. Several approaches will be used to identify which subunit or subunits are essential for expression of signal peptidase activity. Specifically, the active subunit will be crosslinked to a secretory protein precursor containing a photoreactive amino acid analogue. Following subfractionation of the signal peptidase complex, individual subunits will be assayed for signal peptidase activity alone, and in recombination experiments where single subunits are deleted. The mechanism by which ribosomes remain bound to the membrane during nascent chain transport, and then detach following termination of protein synthesis remains largely undefined. Assays for ribosome binding to microsomal membranes using in vitro assembled polysomes will continue to be refined. Detachment of in vitro assembled ribosomes from the membrane will be monitored with a dot-blot hybridization assay using an oligodeoxyribonucleotide probe complementary to wheat germ 5 S rRNA. The long term goals of this research project are the identification and biochemical characterization of function of all necessary components of the endoplasmic reticulum translocation apparatus.